Inhibition of complement C5a receptor protects lung cells and tissues against lipopolysaccharide-induced injury via blocking pyroptosis

W-54011 alleviates LPS-induced lung injury

To investigate the effects of C5a/C5aR inhibition on ALI, we employed an LPS-induced lung injury rat model and applied the C5aR inhibitor W-54011 with doses of 1mg/kg and 5mg/kg. The histological characteristics and the degree of lung injury in the LPS-induced ALI rat model were discovered, as exhibited in Figure 1A, 1B. Compared to the lung tissues of control rats, those of rats undergoing LPS administration showed obvious pathological alterations including intra-alveolar hemorrhage, inter-alveolar septum thickening, pulmonary interstitial edema and inflammatory cells infiltration, indicating the occurrence of ALI. However, with both 1mg/kg and 5mg/kg W-54011 pretreatment, the lung structure nearly returned to the normal appearance and the inflammatory cells infiltration was improved. Meanwhile, rats subjected to LPS displayed elevated Wet/Dry ratios compared to the control rats, which were significantly reduced by W-54011 pretreatment (Figure 1C). These data demonstrated the protective effect of C5aR inhibition on LPS-induced lung damage.

Figure 1. Alterations of histological evaluation, lung wet/dry ratio, BALF cell counts and total protein level. (A) Morphological changes in the lung by H&E staining. The sections were stained with hematoxylin (blue, nucleus) and eosin (pink, muscle fiber). The scale bar represents 50 μm. (B) Morphological changes in the lung by Periodic Acid Schiff (PAS) staining. The sections were stained with hematoxylin (blue, nucleus) and PAS (pink, glycogen). The scale bar represents 50 μm. (C–E) Effect of W-54011 on lung wet/dry ratio (C), BALF cell counts (D) and total protein level (E) 12 h after intratracheal instillation of LPS. n = 7. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.

The BALF of animals was also collected to examine LPS-induced lung injury. LPS triggered a significant increase of cell amount in BALF, whereas W-54011 pretreatment dramatically decreased the count (Figure 1D). In addition, the total protein concentration of BALF can be applied to assess pulmonary capillary permeability, the increase of which could lead to pulmonary edema [21]. Compared with the control group, the total protein concentration of BALF in the LPS group was much higher (Figure 1E). Nevertheless, W-54011 pretreatment reversed the promotion of LPS on total protein concentration of BALF, suggesting that the inhibition of complement C5a/C5aR by W-54011 prevented higher capillary permeability after LPS stimulation (Figure 1E).

W-54011 significantly decreases C5aR influx, levels of inflammatory factors, and cell pyroptosis in LPS-challenged rats

To test the inhibitory efficacy of W-54011 on C5a/C5aR, the content of C5a in serum and level of C5aR in lung tissues were detected. As shown in Figure 2A, 2B, LPS stimulation increased the level of C5a in serum and C5aR in lung tissues. Whereas, W-54011 slightly reduced C5a content and significantly decreased C5aR level, compared with the LPS group. These results implied the potent suppressive effect of W-54011 on the interaction between C5a and its receptor C5aR.

Figure 2. Alterations of C5aR and proinflammatory cytokines level. (A) The content of C5a in serum was measured by ELISA assay. (B) The protein expression of C5aR in lung tissues were detected by western blotting. (C, D) The concentrations of pro-inflammatory cytokines including IL-1β, IL-18, IL-6, TNF-α and HMGB1 in BALF (C) and the lung tissues (D) were measured by ELISA kits, respectively. n = 7. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.

The concentrations of pro-inflammatory cytokines including IL-1β, IL-18, TNF-α, IL-6 and HMGB1 in BALF and lung tissues were presented in Figure 2C, 2D. Results presented that the expressions of IL-1β, IL-18, TNF-α, IL-6 and HMGB1 were distinctly increased in LPS-induced ALI. However, W-54011 pretreatment suppressed the elevated expressions of these pro-inflammatory cytokines in both the BALF and the lung tissues.

TUNEL staining was utilized to detect cell death of lung tissues. Results from Figure 3 revealed that LPS-induced ALI led to an increase in DNA fragmentation, but this change was alleviated by W-54011 addition. Next, the expressions of proteins involved in apoptosis and pyroptosis were tested via western blot. As displayed in Figure 4, the expressions of Bax, cleaved-Caspase 3/11, GSDMD-N, cleaved-Caspase 1, HMGB1, TLR4 and its downstream binding molecule MyD88 were up-regulated, while Bcl-2 was down-regulated in lung tissues after LPS challenge, hinting the activation of apoptosis, pyroptosis and TLR4/MyD88-mediated inflammation upon the stimulation of LPS. Moreover, W-54011 significantly rescued the enhancement of GSDMD-N, cleaved-Caspase 1, HMGB1, TLR4 and MyD88, together with Bax, Bcl-2, and cleaved-Caspase 3/11, compared with LPS group. The results together indicated that the inhibition of C5a/C5aR exerted remarkably effects on apoptosis and pyroptosis pathway.

Figure 3. The effect of W-54011 on TUNEL-positive cells. (A) Representative TUNEL staining for lung tissues. Dead cells were stained with green and DAPI was used for staining the nucleus (blue). The scale bar represents 50 μm. (B) Quantitative analysis for the number of TUNEL-positive cells in different groups (n = 7). ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.

Figure 4. The effect of W-54011 on the expression of proteins related to apoptosis and pyroptosis in lung tissues. (A) The expression of apoptosis-related proteins including Bcl-2, Bax, Caspase 3 and Caspase 11 in different groups was detected using western blotting. (B) The expression of pyroptosis-related proteins including GSDMD, Caspase 1, TLR-4, MyD88 and HMGB1 in different groups was dissected using western blotting. The bar graphs represent the mean gray value of the protein bands after normalized to GAPDH. All data was achieved from 3 times independent replicated experiments. n = 7. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.

W-54011 recovers cell viability, as well as prevents inflammatory response and cell pyroptosis in LPS+ATP-induced cells in the presence of C5a

To further confirm our in vivo findings and the possible mechanism involved in regulation of W-54011, Beas-2B cells were treated with W-54011, C5a or C5a+W-54011 prior to LPS+ATP stimulation. Alterations of cell viability, inflammatory cytokines, apoptosis and pyroptosis-related proteins were observed in cell experiment. In contrast to control cells, LPS+ATP resulted in decreased cell viability, increased cell death and induced inflammatory factors, which were abrogated by W-54011 treatment. Opposite results of C5a treatment were found in contrast with W-54011 treatment in LPS+ATP-treated cells. Besides, the co-treatment of W-54011 could neutralize the actions of C5a (Figure 5).

Figure 5. The effect of W-54011 on cell viability and pro-inflammatory cytokines levels in the presence of C5a in Beas-2B cells. (A) The cell viability in different groups was detected by CCK-8 assay. (B) Representative flow cytometry and quantitative analyses for dead cells. (C) The concentrations of pro-inflammatory cytokines including IL-1β, IL-18, IL-6, TNF-α and HMGB1 in supernatant of Beas-2B cells were determined by ELISA assay. Each experiment was repeated at least 3 times. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.

Results from Figure 6 demonstrated that C5aR level and the expressions of proteins associated with apoptosis and pyroptosis, including Bax, cleaved-Caspase 3/4/5, GSDMD-N, cleaved-Caspase 1, HMGB1, TLR4 and MyD88, were all increased by treatment of LPS+ATP. However, the expressions of these proteins induced by LPS+ATP were weakened in the presence of W-54011 but strengthened by C5a. And the co-treatment of W-54011 also blunted the effects of C5a.

Figure 6. The effect of W-54011 on the expression of proteins related to apoptosis and pyroptosis in Beas-2B cells. (A) The expression of apoptosis-related proteins including Bcl-2, Bax, Caspase 3, Caspase 4 and Caspase 5 in different groups was detected using western blotting. (B) The expression of pyroptosis-related proteins including GSDMD, Caspase 1, TLR-4, MyD88 and HMGB1 in different groups was analyzed using western blotting. (C) The protein expression of C5aR in Beas-2B cells was analyzed using western blotting. Each experiment was repeated at least 3 times. The bar graphs represent the mean gray value of the protein bands after normalized to GAPDH. All data was achieved from 3 times independent replicated experiments. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.

Reagents

Lipopolysaccharide (LPS), W-54011 and ATP were purchased from MedChemExpress (MCE, USA). C5a was purchased from R&D Systems. Zoletil 50 was obtained from Vibac Laboratories (France).

Animals and protocols

The Specific pathogen Free (SPF) adult male Sprague–Dawley (SD) rats were provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All procedures were performed following the Care and Use Guide of Laboratory Animals of the National Institutes of Health, with the approval of the Scientific Investigation Board of Shanghai Jiao Tong University, School of Medicine (SYXK-2018-0027). Forty rats were randomly assigned into four groups: Control, LPS, LPS+W-54011 1mg/kg and LPS+W-54011 5mg/kg. The induction of ALI was operated by intratracheal instillation of LPS as described previously [33, 34]. After anesthetizing via intraperitoneal (i.p.) injection of Zoletil 50 (10 mg/kg), the control animals were instilled intratracheally with 200 μL normal saline, while LPS-treated rats received 50 mg/kg LPS (dissolved in 200 μL saline; Sigma-Aldrich Co, USA) by intratracheal instillation. W-54011 (dissolved in 10% DMSO and then diluted using normal saline; i.p., q.d) was applied to pretreat rats 3 days before LPS instillation. To exclude the actions of DMSO, control and LPS groups also received the equal amount of DMSO. At 12 h after the LPS instillation, rats were anesthetized for next experiments.

Cell culture and treatment

Human bronchial epithelial (Beas-2B) cells (American Type Culture Collection, ATCC) were cultured in 1640 medium (Abcam, UK) with 10% FBS (Wisent) and 1% antibiotics (Thermo Fisher Scientific, USA) in an atmosphere of 5% CO₂ at 37° C. Cells were divided into five groups: Control, LPS+ATP, LPS+ATP+W-54011, LPS+ATP+C5a and LPS+ATP+C5a+W-54011. For C5a and W-54011 treatment, LPS (1 μg/ml) and ATP (5 mmol/L) were added to culture medium after pretreatment with W-54011 (10 μg/ml; dissolved in 10% DMSO and then diluted using PBS) and C5a (50 ng/ml) [35] for 12 h, separately. To exclude the actions of DMSO, control and LPS+ATP groups also treated with the equal amount of DMSO.

CCK-8

For the assessment of cell viability, cells in the 96-well culture dishes after treatment were incubated with 10 μL of WST-8 working solution for 2 h under normal condition, and then the absorbance at 460nm was measured with a microplate reader.

Histological staining

The left lung lobe was fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin, and then cut into 5 μm thick section. After staining with hematoxylin and eosin (H&E) or Periodic Acid Schiff (PAS, Beyotime Biotechnology, China), lung tissue sections were observed with an optical microscope for pathological examination. Besides, some tissue sections were deparaffinized, rehydrated and subjected to TUNEL (Beyotime Institute of Biotechnology, Shanghai, China) staining, and the death of cells was observed under a fluorescence microscope.

Detection of lung wet/dry ratio

After being sacrificed, the lungs of rats were isolated, and the weight of the lungs was immediately recorded as the wet weight. The blood on the lungs was blotted with filter paper, and the lungs were then stored in a 60° C incubator for 48 h. The weight of the lungs was recorded as dry weight. The lung wet/dry weight ratio (W/D) was used to assess the degree of pulmonary edema.

Collection of broncho alveolar lavage fluid (BALF)

Before lavage, rats were anesthetized by injection of Zoletil 50 (10 mg/kg, i.p.). After exposing the chest cavity and intubating the trachea, 5mL PBS was slowly dropped into the lung bronchus 3 times for recovery. The collected solution was centrifuged at 1000 × g for 10 min at 4° C. The supernatant was collected and stored at -80° C for further analysis. The total protein concentration of BALF was determined by BCA method (Beyotime, China), and the sedimented cells were resuspended and subjected to cell counting using a hemocytometer.

ELISA

The activities of inflammatory factors including IL-1β, IL-18, TNF-α, IL-6 and HMGB1 in BALF, lung tissues and cell supernatant, together with C5a levels in serum of rats, were detected by ELISA assay following the instructions (Abcam, Cambridge, UK) as described previously [33]. Results were expressed as optical density (OD) of 450 nm.

Western blotting

The lung tissues were homogenized by the ultrasonication, and total proteins of lung tissues or BEAS-2B cells were extracted using RIPA lysis buffer (Thermo Fisher Scientific) supplemented with PMSF and cocktail (Roche, USA). After quantification, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After being blocked with 5% non-fat milk, the membranes were incubated with the following primary antibodies against Bax, Bcl-2, Caspase 1/3/4/5/11, HMGB1, toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and C5aR. The antibodies were further cultured with HRP-conjugated IgG (Abcam, UK) and visualized by chemiluminescence (Thermo Fisher Scientific). The overall gray value of the protein bands was quantified using Image J software (National Institutes of Health, USA).

Flow cytometry

Cells were subjected to flow cytometry based on Annexin V/PI staining. Briefly, cells were washed with PBS and digested with 0.25% trypsin. Next, FITC-conjugated anti-annexin-V/PI staining antibody (BD Biosciences, USA) and propidium iodide solution were mixed and gently resuspended in Annexin V Binding buffer. After incubation with the cells, flow cytometry was performed using a flow cytometer (Becton Dickinson, USA). The data was analyzed by flow cytometry software.

Statistical analysis

GraphPad Prism 6 software was used for statistical analysis and all data were expressed as mean ± SD. One-way ANOVA was used for mean comparison among groups. P value < 0.05 was considered statistically significant.